Chapter 8 - Adrenal Disorders Saroj Nimkarn, M.D., Asst. Professor of Pediatrics, , Mt. Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029 Maria I. New, M.D., Professor of Pediatrics and Human Genetics, Department of Pediatrics, Mt. Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029 Updated 20 May 2008 TO OBTAIN A DOWNLOAD OF THIS CHAPTER IN WORD OR PDF FORMAT, CLICK HERE	Index Contributors Search

Introduction

Congenital adrenal hyperplasia (CAH) refers to a group of disorders arising from defective cortisol synthesis. The production of cortisol in the zona fasciculata of the adrenal cortex occurs in five major enzyme-mediated steps. Congenital adrenal hyperplasia (CAH) results from deficiency in any one of these enzymes; impaired cortisol synthesis leads to chronic elevations of ACTH and overstimulation of the adrenal cortex resulting in hyperplasia. The five forms of CAH are summarized in Table 1. Impaired enzyme function at each step of adrenal cortisol biosynthesis leads to a unique combination of retained precursors and deficient products. The most common enzyme deficiency that accounts for more than 90% of all CAH cases is 21-hydroxylase deficiency.

EPIDEMIOLOGY

Data from close to 6.5 million newborn screenings worldwide indicate that classical CAH occurs in 1:13,000 to 1:15,000 live births.(2) Nonclassical 21-OHD CAH (NC21-OHD) is more common. The incidence in heterogeneous population of New York City is 1 in 100, making NC21-OHD one of the most frequent recessive disorders in humans.(3) Nonclassical CAH is particularly prevalent in certain ethnicities, showing a high ethnic specificity. NC21-OHD is particularly frequent in Ashkenazi Jews, in whom 1 in 3 are carriers of the allele, and 1 in 27 are affected with NC 21-OHD.(3-5) Steroid 11 -hydroxylase deficiency (11β-OHD) is the second most common cause of CAH, accounting for 5-8% of all cases.(6) It occurs 1 in 100,00 live births in the general population (7, 8) and is more common in some populations of North African origin.(9) In Moroccan Jews, for example the disease incidence was initially estimated to be 1 in 5,000 live births(10); subsequently, it was shown to occur less frequently(11), but remains more common than in other populations. The other forms are considered rare diseases and the incidence is unknown in general population.

PATHOPHYSIOLOGY

Adrenal steroidogenesis occurs in three major pathways: glucocorticoids, mineralocorticoids, and sex steroids as shown in Figure 1. These take place in different areas of the adrenal cortex: glucocorticoids (particularly cortisol), androgens, and estrogens in the zona fasciculata and reticularis; and aldosterone in the zona glomerulosa. An appreciation of these pathways serves as the basis for understanding the different forms of congenital adrenal hyperplasia (CAH). Adrenocorticotropic hormone (ACTH) regulates adrenal steroid production via a rate-limiting step that results in pregnanolone, the principal substrate for the steroidogenic pathway (Figure 1). It promotes StAR protein function in transporting free cholesterol to the inner mitochondrial membrane, the site where a side chain cleavage occurs and the first step in steroidogenesis takes place. The central nervous system controls the secretion of ACTH, its diurnal variation, and its increase during periods of physiological stress via the hypothalamus produced corticotropin-releasing factor (CRF). (12, 13)The hypothalamic-pituitary-adrenal feedback system is mediated through the circulating level of plasma cortisol by negative feedback of cortisol on CRF and ACTH secretion. Therefore, any CAH condition that results in a decrease in cortisol secretion leads to increased ACTH production, which in turn, stimulates (1) excessive synthesis of adrenal products in those pathways unimpaired by the enzyme deficiency and (2) a build up of precursor molecules in pathways blocked by the enzyme deficiency.

The clinical symptoms of the five different forms of CAH result from the particular hormones that are deficient and those that are produced in excess as outlined in table 1. In the most common form 21 OHD-CAH, the function of 21-hydroxylating cytochrome 450 is inadequate creating a block in cortisol production pathway. This leads to an accumulation of 17-hydroxyprogesterone (17-OHP), a precursor adjacent to the 21-hydroxylation step. Excess 17-OHP is then shunted into the intact androgen pathway, where the 17,20-lyase enzyme converts the 17-OHP to ⁴-androstenedione, which is converted into androgens. Since the mineralocorticoid pathway requires minimal 21-hydroxylase activity, mineralocorticoid deficiency is a feature of the most severe form of the disease called salt wasting CAH. The enzyme defect in the nonclassical form of 21-OHD CAH is only partial so that there are no apparent clinical cortisol deficiency and salt wasting in this mild form of 21-OHD CAH. The analogy of all other enzyme deficiencies in term of precursor retention and product deficiencies are shown in Table 1.

A very rare form of CAH not included in this table is Cytochrome P450 oxidoreductase deficiency (POR gene defect). It is characterized by an apparent combined P450C17 (17-hydroxylase) and P450C21 (21-hydroxylase) deficiency. Affected girls are born with ambiguous genitalia, indicating intrauterine androgen excess. Virilization does not progress after birth. The 17-OH progesterone levels are elevated, as in 21-hydroxylase deficiency, while androgen levels are low; cortisol may be normal but is poorly responsive to adrenocorticotropic hormone. Conversely, affected boys are sometimes born undermasculinized. Boys and girls can also present with bone malformations, in some cases resembling a pattern seen in patients with Antley-Bixler syndrome. Findings of biochemical investigations of urinary steroid excretion in affected patients have shown the accumulation of steroid metabolites, indicating impaired C17 and C21 hydroxylation.

CLINICAL FEATURES

External genitalia

Virilizing forms of CAH; Classical 21-OHD CAH and 11β-hydroxylase deficiency

Females with Classical 21-OHD and 11β-hydroxylase deficiency CAH generally present at birth with virilization of their genitalia. Adrenocortical function begins around the 7^th week of gestation(14); thus, a female fetus with classical CAH is exposed to adrenal androgens at the critical time of sexual differentiation (approximately 9 to 15 weeks gestational age). The degree of genital virilization may range from mild clitoral enlargement alone to, in rare cases, a penile urethra. Degrees of genital virilization are classified into five Prader stages (15) (see Figure 2):

Internal genitalia

As the androgens interact with the receptors on genital skin, they induce changes in the developing external female genitalia such as clitoral enlargement, fusion of the labial folds, and rostral migration of the urethral/vaginal perineal orifice. In contrast, internal female genitalia (the uterus, fallopian tubes and ovaries) develop normally because females with CAH have normal ovaries and do not produce anti- müllerian hormone (AMH). These müllerian derivatives are not androgen responsive. Therefore, the affected female who is born with virilized external genitalia will have a normal uterus, normal fallopian tubes, and normal ovaries. Females with classical CAH maintain the internal genitalia potential for fertility.

Postnatal effects and growth

Lack of postnatal treatment in boys and girls results in continued exposure to excessive androgens, causing progressive penile or clitoral enlargement, the development of premature pubic (pubarche) and axillary hair, and acne. Advanced somatic and epiphyseal development occurs with exaggerated growth during childhood. However, this rapid linear growth is usually accompanied by premature epiphyseal maturation and closure, resulting in a final adult height that is typically below that expected from parental heights (on average 9 cm below the mid-parental target height)(16). On the other hand, poor growth can occur in patients with 21-OHD as a result of glucocorticoid treatment when replacement therapy exceeds physiological requirements.

Final height in classical 21-OHD (as well as nonclassical 21-OHD) is one of the features least amenable to glucocorticoid therapy even among patients with excellent adrenal control. A study of growth hormone therapy alone or in combination with a GnRH analog in CAH patients with compromised height prediction showed improvement in short and long term growth to reduce the height deficit. (16, 17)

Puberty

In the majority of patients treated adequately from early life, the onset of puberty in both girls and boys with classical 21-OHD occurs at the expected chronological age.(18, 19) However, recent careful study showed that the mean ages at onset of puberty in both males and females were somewhat younger than the general population, but did not differ significantly among the three forms of 21-OHD. (20)

In those who are inadequately treated, advanced epiphyseal development results, which can lead to central precocious puberty. In those with advanced body maturation at the initial presentation, such as simple virilizing males, the exposure to elevated androgens followed by the sudden decreased androgen levels after initiation of glucocorticoid treatment may cause an early activation of the hypothalamic-pituitary-gonadal axis. Studies suggest that excess adrenal androgens (aromatized to estrogens) inhibit the pubertal pattern of gonadotropin secretion by the hypothalamic-pituitary axis.(21) This inhibition which probably takes place via a negative feedback effect can be reversed by glucocorticoid treatment.

Following the onset of puberty, in a majority of successfully treated patients, the milestones of further development of secondary sex characteristics in general appear to be normal.(18-20) In adolescents and adults, signs of hyperandrogenism may include male-pattern alopecia (temporal balding), acne, and in females, hirsutism.

Gender role behavior

Prenatal androgen exposure in females affected with classic forms of 21-OHD CAH has a virilizing effect on childhood behavior, as well as the development of external genitalia. Both physical and behavioral masculinization were related to each other and to genotype, indicating that behavioral masculinization in childhood is a consequence of prenatal androgen exposure. Further, changes in childhood play behavior correlated with reduced female gender satisfaction and reduced heterosexual interest in adulthood. Prenatal androgen exposure is related to a decrease in self-reported femininity in dose response manner in adulthood but not the degree of masculinity. Affected adult females are more likely to have gender dysphoria, and experience less heterosexual interest and reduced satisfaction with the assignment to the female sex. With regards to cognitive abilities, such as visuospatial/motor ability and handedness, the effect of prenatal androgen exposure remains unclear. Results from different studies are controversial. Salt wasting patients were found to have lower intelligence (as determined by full-scale IQ scores) than simple virilizing and nonclassic patients. In contrast to females, males affected with CAH do not show a general alteration in childhood play behavior, core gender identity and sexual orientation.

Fertility

Difficulty with fertility in females with CAH may arise for various reasons, including anovulation, secondary polycystic ovarian syndrome, irregular menses, non- suppressible serum progesterone levels, or an inadequate introitus. Fertility is reduced in salt-wasting 21-OHD with rare reports of pregnancy. In a retrospective survey of fertility rates in a large group of females with classical CAH, simple virilizers were shown to be more likely to become pregnant and carry the pregnancy to term.(22) Adequate glucocorticoid therapy is probably an important variable with respect to fertility outcome. The development of PCOS in CAH patients is not uncommon and may be related to both prenatal and postnatal excess androgen exposure, which can affect the hypothalamic-pituitary-gonadal axis. Males with CAH, particularly if poorly treated, may have reduced sperm counts (23, 24) and low testosterone as a result of small testes due to suppression of gonadotropins and sometimes intra-testicular adrenal rests. All of these complications may result in diminished fertility.

In male patients with classical CAH, several long-term studies indicate that those who have been adequately treated undergo normal pubertal development, have normal testicular function, and normal spermatogenesis and fertility. (20, 25-27)However, small testes and aspermia can occur in patients as a result of inadequately controlled disease. (28-30)Testicular adrenal rest tumor can lead to end stage damage of testicular peranchyma, most probably as a result of longstanding obstruction of the seminiferous tubules(31). In contrast, some investigators have reported normal testicular maturation as well as normal spermatogenesis and fertility in patients who had never received glucocorticoid treatment. (25, 27, 32)

A complication in postpubertal boys with inadequate control of CAH is hyperplastic nodular testes. Almost all these patients were found to have adenomatous adrenal rests within the testicular tissue, as indicated by the presence of specific 11β-hydroxylated steroids in the blood from gonadal veins. (33)These tumors have been reported to be ACTH dependent and to regress following adequate steroid therapy.(34-38) These testicular adrenal rests are more frequent in males with salt-wasting CAH and are associated with an increased risk of infertility. (23, 39)

Salt wasting in 21-OHD

When the loss of 21-hydroxylase function is severe, adrenal aldosterone secretion is not sufficient for sodium reabsorption by the distal renal tubules, and individuals suffer from salt wasting as well as cortisol deficiency and androgen excess. Infants with renal salt wasting have poor feeding, weight loss, failure to thrive, vomiting, dehydration, hypotension, hyponatremia, and hyperkalemic metabolic acidosis progressing to adrenal crisis (azotemia, vascular collapse, shock, and death). Adrenal crisis can occur as early as age one to four weeks. The salt wasting is presumed to result from inadequate secretion of salt-retaining steroids, primarily aldosterone. In addition, hormonal precursors of the 21-OH enzyme may act as antagonists to mineralocorticoid action in the sodium-conserving mechanism of the immature newborn renal tubule. (40-42)

Affected males who are not detected in a newborn screening program are at high risk for a salt-wasting adrenal crisis because their normal male genitalia do not alert medical professionals to their condition; they are often discharged from the hospital after birth without diagnosis and experience a salt-wasting crisis at home. On the other hand, salt wasting females are born with ambiguous genitalia that trigger the diagnostic process and appropriate treatment. It is important to recognize that the extent of virilism may not differ among the two classical CAH, simple virilizing and salt-wasting form. Thus, even a mildly virilized newborn with 21-OHD should be observed carefully for signs of a potentially life-threatening crisis within the first few weeks of life.

It has been observed that an aldosterone biosynthetic defect apparent in infancy may be ameliorated with age. (43, 44)Speiser et al. reported the spontaneous partial recovery in adulthood of a patient with documented severe salt wasting in infancy.(45) Therefore, it is desirable to follow the sodium and mineralocorticoid requirements carefully by measuring PRA in patients who have been diagnosed in the neonatal period as salt wasters.

Nonclassical 21-OHD

Nonclassical 21-OHD (NC 21-OHD), also known as late-onset 21-OHD, is much more common than the classical form, with an incidence as high as 1:27 in Ashkenazi Jews.(3) Individuals with the non-classical (NC) form of 21-OHD have only mild to moderate enzyme deficiency and present postnatally with signs of hyperandrogenism. By definition, females with NC-CAH do not have virilized genitalia at birth.

NC-CAH may present at any age after birth with a variety of hyperandrogenic symptoms, excluding ambiguous genitalia. This form of CAH results from a mild deficiency of the 21-hydroxylase enzyme. Table 2 summarizes main clinical characteristics of all forms of 21 OHD CAH. Similar to classical CAH, NC-CAH may cause premature development of pubic hair, advanced bone age and accelerated linear growth velocity in both males and females. Severe cystic acne has also been attributed to NC-CAH.

Women may present with symptoms of androgen excess, including hirsutism, temporal baldness, and infertility. Menarche in females may be normal or delayed, and secondary amenorrhea is a frequent occurrence. Further virilization may include hirsutism, male habitus, deepening of the voice, or male-pattern alopecia (temporal recession). Polycystic ovarian syndrome may also be seen as a secondary complication in these patients. Possible reasons for the development of PCOS include reprogramming of the hypothalamic-pituitary-gonadal axis from prenatal exposure to androgens, or chronic levels of excess adrenal androgens that disrupt gonadotropin release and have direct effects on the ovary, ultimately leading to the formation of cysts. Because of the overlap of hyperandrogenic symptoms, it is important to consider NC 21-OHD in a patient diagnosed with PCO.

In adult males, early balding, acne, or infertility may prompt the diagnosis of NC-CAH. A highly reliable constellation of physical signs of adrenal (as opposed to testicular) androgen excess is the presence of pubic hair, enlarged phallus, and relatively small testes. They may have small testes compared to the phallus that results from suppression of the hypothalamic-pituitary-gonadal axis from adrenal androgens. They may also develop intra-testicular adrenal rests, which can cause infertility, although some untreated men have been fertile(23). Symptoms in adult males with NC-CAH may be limited to short stature or oligozoospermia and diminished fertility.

A subset of individuals with NC-21OHD are completely asymptomatic when detected (usually as part of a family study), but it is thought, based on longitudinal follow-up of such patients, that symptoms of hyperandrogenism may wax and wane with time. The presence of 21-OHD can also be discovered during the evaluation of an incidental adrenal mass.(46) An increased incidence of adrenal incidentalomas has been found, reported as high as 82% in patients with 21-OHD and up to 45% in subjects heterozygous for 21-OHD mutations. This probably arises from hyperplastic tissue areas and does not require surgical intervention. (47)

Other forms of congenital adrenal hyperplasia

11β-hydroxylase deficiency

Virilization and low renin hypertension are the prominent clinical features of 11β hydroxylase deficiency(48). The virilization signs and symptoms of this disorder are similar to classical 21-OHD. Despite failure of aldosterone production, overproduction of DOC, in vivo a less potent mineralocorticoid, causes salt retention and hypertension. Elevated blood pressure is usually not identified until later in childhood or in adolescence, although its appearance in an infant 3 months of age has been documented(49). In addition, hypertension correlates variably with biochemical values, and clinical signs of mineralocortiocid excess and the degree of virilization are not well correlated. Some severely virilized females are normotensive, whereas mildly virilized patients may experience severe hypertension leading to fatal vascular accidents(50). Complications of long standing uncontrolled hypertension, including cardiomyopathy, retinal vein occlusion and blindness have been reported in 11β-OHD patients(51, 52). Potassium depletion develops concomitantly with sodium retention, but hypokalemia is variable. Renin production is suppressed secondary to mineralocortiocid -induced sodium retention and volume expansion. Aldosterone production is low secondary to low serum potassium and low plasma renin. (See chapter on Endocrine Hypertension in Childhood).

3β-hydroxysteroid dehydrogenase deficiency

There are two forms of the 3β-hydroxysteroid dehydrogenase enzyme (3β-HSD), types I and II. Type II 3β-HSD enzyme is expressed in the adrenal cortex and gonads and is responsible for conversion of Delta 5 (D5) to Delta 4 (D4) steroids. The term delta indicates which carbon position in steroid skeleton has double bond. This enzyme is essential for the formation of progesterone, the precursor for aldosterone, 17-OHP, the precursor for cortisol in the adrenal cortex, as well as androstenedione, testosterone, and estrogen in the adrenal cortex and gonads.(53, 54) Therefore, deficiency of 3ß-HSD in the classic form of 3ß-HSD deficiency CAH results in insufficient cortisol synthesis, salt wasting in the most severe form, and virilization of external genitalia in females due to androgen effect from the peripheral conversion of circulating D5 precursors to active D4 steroids. Simultaneous type II 3ß-HSD deficiency in the gonads results in incomplete virilization of the external genitalia in males. Thus, genital ambiguity can result in both sexes.

17α-hydroxylase/17,20 lyase deficiency

Steroid 17α-hydroxylase/17,20 lyase deficiency accounts for approximately 1% of all CAH cases and affects steroid synthesis in both the adrenals and gonads. (55) Patients have impaired cortisol synthesis, leading to ACTH oversecretion, which increases serum levels of deoxycorticosterone and especially corticosterone, resulting in low renin hypertension, hypokalemia, and metabolic alkalosis. Affected females are born with normal external genitalia. Affected males are also born with under-virilized genitalia due to their deficient gonadal testosterone production. 17α-Hydroxylase/17,20 lyase deficiency is often recognized at puberty in female patients who fail to develop secondary sex characteristics.

Lipoid hyperplasia

Congenital lipoid hyperplasia is an extremely rare and severe form of CAH in which cholesterol is not converted to pregnenolone, resulting in deficient synthesis of all adrenal and gonadal steroids. The failure to convert cholesterol to pregnenolone leads to accumulations of cholesterol and cholesterol esters in the newborn. Recent studies have revealed that abnormalities of the steroidogenic acute regulatory (StAR) protein are responsible for this disorder.(56, 57) StAR is involved in the transfer of cholesterol from the outer to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis. Males with congenital lipoid hyperplasia are born with female-appearing external genitalia. Females have a normal genital phenotype at birth but remain sexually infantile without treatment. Salt wasting occurs in both males and females. If not detected and treated, lipoid CAH is usually fatal. (58)

Cytochrome P450 oxidoreductase deficiency

Cytochrome P450 oxioreductase deficiency is another rare form of CAH that is caused by mutations in the POR gene. It is characterized by an apparent combined P450C17 (17-hydroxylase) and P450C21 (21-hydroxylase) deficiency. POR is the electron donor for all microsomal P450 enzymes, including the three steroidogenic enzymes P450c17 (17-hydroxylase/17,20-lyase), P450c21 (21-hydroxylase), and P450aro (aromatase).. Affected girls are born with ambiguous genitalia, indicating intrauterine androgen excess. Virilization does not progress after birth. The 17-OH progesterone levels are elevated, as in 21-hydroxylase deficiency, while androgen levels are low; cortisol may be normal but is poorly responsive to adrenocorticotropic hormone. Conversely, affected boys are sometimes born undermasculinized. Boys and girls can also present with bone malformations, in some cases resembling a pattern seen in patients with Antley-Bixler syndrome. Findings of biochemical investigations of urinary steroid excretion in affected patients have shown the accumulation of steroid metabolites, indicating impaired C17 and C21 hydroxylation. This suggests concurrent partial deficiencies of the 2 steroidogenic enzymes, P450C17 and P450C21.

GENETICS

In general, all forms of CAH are transmitted in autosomal recessive mode of inheritance as a single gene disorder. However, there have been reports of cases where none or only one mutation in the responsible gene was identified : for example in 21 OHD CAH(59, 60); P450SCC mutation in congenital lipoid adrenal hyperplasia (61) and POR deficiency(62). The genes responsible for each form of CAH are shown in Table 3.

CYP21A2

Hormonally and clinically defined forms of 21-OHD CAH are associated with distinct genotypes characterized by varying enzyme activity demonstrated through in vitro expression studies. The gene encoding 21-hydroxylase is a microsomal cytochrome P450 termed cytochrome P450, family 21, subfamily A, polypeptide 21 (CYP21A2(63); previously called P450c21B, CYP21B or CYP21 (Online Mendelian Inheritance in Man [OMIM] database number 201910) located on the short arm of chromosome 6 within the human leukocyte antigen (HLA) complex. (64)CYP21A2 and its homolog, the pseudogene CYP21A1P (previously called CYP21P, CYP21A), alternate with two genes, C4B and C4A, that encode the two isoforms of the fourth component of the serum complement system. (65)The protein-encoding sequence of CYP21P is 98% homologous to that of CYP21A2. The high degree of homology permits two types of mutation causing recombination events: (1) unequal crossing over during meiosis that results in complementary deletions/duplications of CYP21, (65, 66)and (2) non-correspondences between the pseudogene and the coding gene that, if transferred by gene conversion, result in deleterious mutations. (67)

More than 100 mutations have been described including point mutations, small deletions, small insertions and complex rearrangements of the gene. (68)Figure 3 demonstrates the common mutations in CYP21A2 and their related phenotypes. In recessive disorders, the less severe mutation of the two alleles typically dictates phenotype. Classical 21-OHD is most often caused by two alleles with severe mutations. In contrast to the classical form, patients with nonclassical 21-OHD are predicted to have mild mutations on both alleles or one severe and one mild mutation (compound heterozygosity) of CYP21A2. It is not always possible, however, to accurately predict the phenotype on the basis of the genotype—such predictions have been shown to be 79–88% accurate. (59, 69) with some nonconcordance .

DIAGNOSIS

HORMONAL DIAGNOSIS

Potential diagnosis of CAH must be suspected in infants born with ambiguous genitalia. The physician is obliged to make the diagnosis as quickly as possible, to initiate therapy, and to arrest the effects of the enzyme disorders. The diagnosis and rational decision of sex assignment must rely on the determination of genetic sex, the hormonal determination of the specific deficient enzyme, and an assessment of the patient's potential for future sexual activity and fertility. Physicians are urged to recognize the physical characteristics of CAH in newborns (e.g. ambiguous genitalia) and to refer such cases to appropriate clinics for full endocrine evaluation. As indicated in Table 1, each form of CAH has its own unique hormonal profile, consisting of elevated levels of precursors and elevated or diminished levels of adrenal steroid products. Traditionally, laboratories measured urinary excretion of adrenal hormones or their urinary metabolites (e.g. 17-ketosteroids). However, collection of 24 h urine excretion is difficult, particularly in neonates.(70) Therefore, simple and reliable radioimmunoassays are utilized now for measuring circulating serum levels of adrenal steroids.(71) Alternatively, a noninvasive random urine collection in the first days of life for steroid hormone metabolites and precursor/product ratio assessments can be measured simultaneously. It can be used independently or in conjunction with serum steroid assays to increase accuracy and confidence in making diagnosis and distinguishing the separate enzymatic forms of the disorder. (72)

Diagnosis of the 21-OHD CAH can also be confirmed biochemically by hormonal evaluation. In a randomly timed blood sample, a very high concentration of 17-hydroxyprogesterone (17-OHP), the precursor of the defective enzyme, is diagnostic of classical 21-OHD. Such testing is the basis of the newborn-screening program developed to identify classically affected patients who are at risk for salt wasting crisis.(73) Only 20µl blood, obtained by heel prick and blotted on microfilter paper, is used for this purpose to provide a reliable diagnostic measurement of l7-OHP. The simplicity of the test and the ease of transporting microfilter paper specimens through the mail has facilitated the implementation of CAH newborn screening programs worldwide. The universal newborn screening program has been implemented in 49 out of 51states in the United States. The states that have mandated newborn screening for 21-OHD (CAH) are identified in the National Newborn Screening Status Report (PDF). False-positive results are, however, common with premature infants. Appropriate references based on weight and gestational age are therefore in place in many screening programs. (74, 75)The majority of screening programs use a single screening test without retesting of questionable 17-OHP concentrations. To improve efficacy, a small number of programs perform a second screening test of the initial sample to re-evaluate borderline cases identified by the first screening. Current immunoassay methods used in newborn screening programs yield a high false positive rate. In order to decrease this high rate, liquid-chromatography- tandem mass spectrometry measuring different hormones (17-OHP, Δ4-androstenedione and cortisol) has been suggested as a second-tier method of analyzing positive results.

The gold standard for hormonal diagnosis is the corticotropin stimulation test (250 μg cosyntropin intravenously), measuring levels of 17-OHP and Δ4 androstenedione at baseline and 60 min. These values can then be plotted in the published nomogram to ascertain disease severity. (76, 77) (figure 4) It is important to note that the corticotropin stimulation test should not be performed during the initial 24 hours of life as samples from this period are typically elevated in all infants and may yield false-positive results. The corticotropin stimulation test is crucial in establishing hormonal diagnosis of nonclassical form of the disease since early-morning values of 17-OHP may not be sufficiently elevated to allow accurate diagnosis.

Prenatal Diagnosis and Prenatal Treatment

Prenatal Diagnosis of 21OHD

A number of approaches to prenatal identification of affected fetuses have been used. In 1965, Jeffcoate et al (78) first reported a successful prenatal diagnosis of 21OHD, based on elevated levels of 17-ketosteroids and pregnanetriol in the amniotic fluid. The hormonal diagnostic test for 21OHD is amniotic fluid 17-OHP. Androstenedione Δ⁴, may also be employed as an adjunctive diagnostic assay(79). Hormonal diagnosis is currently only used when molecular diagnosis is unavailable.

Recent advances in genotyping of the CYP21A2 gene have made molecular genetic studies of extracted fetal DNA the ideal method to diagnose 21OHD CAH in the fetus(80). Approximately 95% to 98% of the mutations causing 21OHD have been identified through a combination of molecular genetic techniques to study large gene rearrangement, and arrays of point mutations(81-83). CVS, rather than amniocentesis, with molecular genotyping is the preferred diagnostic method in use. Chorionic villus sampling is performed at the 10^th to 11^th week of gestation, while amniocentesis is usually performed in the second trimester. As we only wish to treat affected females till term and only ¼ of the fetuses will be affected and ½ will be males, 7 out of 8 fetuses do not require treatment. Thus, amniocentesis, which is performed later in gestation, results in treatment of unaffected fetuses for a longer period of time than CVS. However, amniocentesis can be used as a reliable alternative method of prenatal diagnosis when CVS in unavailable. In such instances, the supernatant is used for hormonal measurement and the cells are cultured to obtain a genotype through DNA analysis. The supernatant hormone measurements distinguish affected status from non affected status only in SW patients. Nonetheless, pitfalls do occur in a small percentage of the patients undergoing prenatal diagnosis utilizing genetic diagnosis, such as undetectable mutations(84), allele drop out(85), or maternal DNA contamination. Determination of satellite markers may increase the accuracy of molecular genetic analysis(86).

Studying fetal DNA from maternal plasma may reduce the need for invasive procedures to obtain fetal samples in at-risk pregnancies if analysis of the fetal DNA becomes possible at the optimal time in gestation. Reports of sex determination from fetal DNA in maternal plasma show promise(87, 88), but require more confirmation of successful results.

Preimplantation diagnosis

Preimplantation genetic diagnosis (PGD) identifies genetic abnormalities in preimplantation embryos prior to embryo transfer, so only unaffected embryos established from IVF are transferred. The procedure has been utilized in many monogenic recessive disorders such as cystic fibrosis, hemoglobinopathies, spinal muscular atrophy and Tay Sach’s disease. PGD is being used for a growing number of genetic diseases(89). Preimplantation diagnosis has not been utilized in CAH except for one report which did not result in a pregnancy(89). It would be desirable to have further studies of preimplantation diagnosis in CAH families.

Prenatal Treatment

In 21OHD, prenatal treatment with dexamethasone has been used since 1984(90). Institution of therapy before the 9^th week of gestation, prior to the onset of adrenal androgen secretion, effectively suppresses excessive adrenal androgen production and prevents virilization of external female genitalia. Dexamethasone is used because it binds minimally to cortisol binding globulin (CBG) in the maternal blood, and unlike hydrocortisone, escapes inactivation by placental 11 -hydroxysteroid dehydrogenase enzyme. Thus, dexamethasone crosses the placenta from the mother to the fetus and suppresses ACTH secretion with longer half life compared to other synthetic steroids(91).

When dexamethasone administration begins as early as the 8^th week of gestation, the treatment is blind to the disease status and sex of the fetus. If the fetus is later determined upon karyotype to be a male, or an unaffected female upon DNA analysis, treatment is discontinued. Otherwise, treatment is continued to term(79). A simplified algorithm of management of potentially affected pregnancies is shown in Figure 4. The optimal dosage and timing is 20 µg/kg/day of dexamethasone per maternal pre-pregnancy body weight, in three divided doses, starting as soon as pregnancy is confirmed, and no later than 9 weeks after the last menstrual period(92, 93). The mother’s blood pressure, weight, glycosuria, HbA_1C, symptoms of edema, straie and other possible adverse effects of dexamethasone treatment should be carefully observed throughout pregnancy. Urinary estriol may be monitored in the mother after 15 to 20 weeks of gestation to indicate fetal adrenal suppression, and to assure compliance (94).

Figure 5 Simplified algorithm of treatment, diagnosis and decision-making for prenatal treatment of fetuses at risk for 21-hydroxylase deficiency congenital adrenal hyperplasia

OUTCOME OF PRENATAL TREATMENT

Not only does prenatal treatment effectively minimize the degree of female genital virilization in the patients, it also lessens the high-level androgen exposure of the brain during differentiation. The latter is thought to cause a higher tendency to gender ambiguity in some females with CAH. (95, 96)Genital virilization in female newborns with classical 21-OHD CAH has significant potential adverse psychosocial implications that may be greatly alleviated by prenatal treatment. Couples who are both carriers for severe 21-hydroxylase mutations experience anxiety about having an affected, virilized daughter. Parents may have many concerns about the life of a virilized female: that she may not be able to marry, have normal sex function, or have children. Concerns also arise about the potentially extensive surgery needed to correct the child’s genitalia to a normal appearance.

Although some uncertainties and concerns have been raised about the long-term safety of prenatal diagnosis and treatment, (97, 98)compelling data from large cohorts of pregnancies with prenatal diagnosis and treatment of 21-OHD CAH(99, 100) prove its efficacy and safety. In our studies, (101, 102)all the mothers who received prenatal treatment (partial-term or full-term) stated that they would take dexamethasone again for a future pregnancy. Rare adverse events have been reported in treated children, but no harmful effects have been documented that can be clearly attributed to the treatment.(103) Another long-term follow-up study in Scandinavia (104) showed that 44 children who were variably treated prenatally demonstrated normal prenatal and postnatal growth compared to matched controls. Further, there was no observed increase in fetal abnormalities or fetal death. Although some abnormalities in postnatal growth and behavior were observed among dexamethasone exposed offsprings but none could logically be explained by the present knowledge of teratogenic effects of glucocorticoids.

In our comprehensive studies of a total of almost 600 pregnancies, (99, 102)80 of whom were prenatally treated until term and 27 who were male and received dexamethasone for a short period of time, the newborns in the dexamethasone treated group did not differ in weight, length or head circumference from untreated, unaffected siblings. No significant or enduring side effects were noted in either the mothers or the fetuses. Greater weight gain in treated versus untreated mothers did occur, as well as the presence of striae and edema. Excessive weight gain was lost after birth. No differences were found regarding gestational diabetes or hypertension. No cases have been reported of cleft palate, placental degeneration or fetal death, which have been observed in the rodent model of in utero exposure to high-dose glucocorticoids. (105)One explanation for the safety of human versus rodent is that glucocorticoid receptor-ligand systems in human differ from that of rodents (106, 107) The incidence of fetal deaths in treated pregnancies does not exceed that predicted for the general population.

Concerns regarding glucocorticoid effects to fetal brain arise from studies of other conditions rather than direct studies on prenatal treatment of 21 OHD CAH. These include studies whereby much higher doses of dexamethasone were given to the human subjects at the later part of pregnancy (108) or to animals (109, 110) and therefore hold little relevance to using dexmethasone prenatally in CAH. Lack of current long term outcome studies also makes prenatal dexamethasone treatment a subject of intense debate(103). Most experts in centers around the world agree that there is definitely a need for long term follow up studies of both short and long term treated children in systematic fashion including psychological/ behavioral and somatic effects.(111)

One of the concerns about prenatal treatment relate to the psychological development of affected individuals. Long-term studies on the psychological development of patients treated prenatally are currently underway to shed light on possible subtle effects of glucocorticoids that might go unnoticed during early life. (112) A preliminary report of a small pilot study of 26 prenatally treated children compared to controls found no negative effects of dexamethasone on developmental milestones or cognitive development. The pilot study did find increased internalizing behavioral traits, such as shyness, in the children prenatally treated with dexamethasone,(113) contrary to more-aggressive behavior found in females with untreated 21-OHD CAH. (114) Some of these psychobiological studies utilizing questionnaire methods may have limitations in delineating subtle effects. However, the study claimed results needed confirmation as numbers were very small. Batteries of diversely constructed assessment instruments have been developed and been rigorously evaluated(115). Large- scale, international studies of cognitive function and psychological outcomes utilizing standardized instruments in behavioral medicine, as well as growth and metabolic effects in the mother and offspring have been initiated.

Prenatal diagnosis and treatment of 11β-OHD CAH

A number of approaches to prenatal identification by measuring steroid precursors in affected fetuses have been used (116-118) . Recent advances in genotyping of the CYP11B1 gene have made molecular genetic studies of extracted fetal DNA, the ideal method to diagnose 11β-OHD CAH in the fetus (7, 119, 120). The established protocol of prenatal diagnosis and treatment in 21OHD CAH can be applied to 11β-OHD CAH. Successful results in prenatal diagnosis and treatment in 11β-OHD CAH have been reported (119, 121).

TREATMENT

Hormone Replacement

The goal of therapy in CAH is to both correct the deficiency in cortisol secretion and to suppress ACTH overproduction. Proper treatment with glucocorticoid reduces stimulation of the androgen pathway, thus preventing further virilization and allowing normal growth and development. The usual requirement of hydrocortisone (or its equivalent) for the treatment of classical CAH is about 10-15 mg/m²/day divided into 2 or 3 doses per day. Dosage requirements for patients with NC-21OHD CAH may be less. Adults may be treated with the longer-acting dexamethasone or prednisone, alone or in combination with hydrocortisone. A small dose of dexamethasone at bedtime (0.25 to 0.5 mg) is usually adequate for androgen suppression in non-classical patients. Anti-androgen treatment may be useful as adjunctive therapy in adult women who continue to have hyperandrogenic signs despite good adrenal suppression. Females with concomitant PCOS may benefit from an oral contraceptive, though this treatment would not be appropriate for patients trying to get pregnant. Treatment of adult males with NC-21 OHD may not be necessary, though our group has found that it may be helpful in preventing adrenal rest tumors and preserving fertility. Titration of the dose should be aimed at maintaining androgen levels at age and sex-appropriate levels and 17-OHP levels of <1000 ng/dL. Concurrently, over-treatment should be avoided because it can lead to Cushing syndrome. Depending on the degree of stress, stress dose coverage may require doses of up to 50-100 mg/m2/day.

Patients with salt wasting CAH have elevated plasma renin in response to the sodium-deficient state, and they require treatment with the salt-retaining steroid 9 -fludrocortisone acetate. Although patients with the SV and NC forms of CAH can make adequate aldosterone, the aldosterone to renin ratio (ARR) has been found to be lower than normal, though not to the degree seen in the salt-wasting form(122). It has not been customary to supplement conventional glucocorticoid replacement therapy with the administration of salt-retaining steroids in the SV and NC forms of CAH, though there has been some suggestion that adding fludrocortisone to patients with elevated PRA may improve hormonal control of the disease(117). The requirement for fludrocortisone appears to diminish with age, and over-suppression of the PRA should be avoided, to prevent complications from hypertension and excessive mineralocorticoid activity.

In non-life-threatening periods of illness or physiologic stress, the corticosteroid dose should be increased to 2 or 3 times the maintenance dose for the duration of that period. Each family should be given injection kits of hydrocortisone for emergency use (25 mg for infants, 50 mg for children, and 100 mg for adults). In the event of a surgical procedure, a total of 5 to 10 times the daily maintenance dose may be required during the first 24-48 hours, which can then be tapered over the following days to the normal preoperative schedule. Stress doses of dexamethasone should not be given because of the delayed onset of action. It is not necessary for increased mineralocorticoid doses during these periods of stress.

It is imperative for all patients who are receiving corticosteroid replacement therapy such as these patients with CAH to wear a Medic-Alert or other identifying bracelet that will enable correct and appropriate therapy in case of emergencies. Additionally, all responsible family members should be trained in the intramuscular administration of hydrocortisone.

Bone Mineral Density

In order to adequately suppress androgen production in patients with CAH, the usual requirement of hydrocortisone is generally higher than the endogenous secretory rate of cortisol. Chronic therapy with glucocorticoids at supraphysiologic levels can result in diminished bone accrual and lead to osteopenia and osteoporosis. Glucocorticoid induced bone loss is a well-known phenomenon and is the most prevalent form of secondary osteoporosis (123-126).

Unlike other diseases treated with chronic glucocorticoid therapy, however, the effect of glucocorticoid replacement in CAH on BMD is unclear. Previous studies of patients with 21OHD have reported increased, normal, or decreased BMD(127-133). It has been postulated that the elevated androgens typically found in patients may have a protective effect on bone integrity, but the precise mechanism is unknown. The increased adrenal androgens, which are converted to estrogens, may counteract the detrimental effects of GCs on bone mass. This may explain why older CAH women, particularly those who are post-menopausal, are at higher risk for osteoporosis than younger CAH patients. A study is currently underway to examine a potential mechanism by which certain CAH patients are protected against glucocorticoid induced bone loss.

Monitoring of therapy

Optimal corticosteroid therapy is determined by adequate suppression of adrenal hormones balanced against normal physiological parameters. The goal of corticosteroid therapy is to give the lowest dose required for optimal control. Adequate biochemical control is assessed by measuring serum levels 17-OHP and androstenedione; serum testosterone can be used in females and prepubertal males (but not in newborn males). We recommend that hormone levels are measured at a consistent time in relation to medication dosing, usually 2 hours after the morning corticosteroid dose with a target 17-OHP range between 100 and 1000 ng/dL. Measurement of plasma renin is used to monitor the efficacy of mineralocorticoid therapy in patients with salt wasting form.

Surgery

In the past, it was routine to recommend early corrective surgery for neonates born with ambiguous genitalia. However, in recent years, the implementation of early corrective surgery has become increasingly controversial due to lack of data on long-term functional outcome. (100) Because of the scarcity of this data, the role of the parents in sex assignment becomes crucial in all aspects of the decision making process, and should include full discussion of the controversy and all possible therapeutic options for the intersex child, particularly early versus delayed surgery. Furthermore, a multidisciplinary case-by-case approach, involving pediatric endocrinology, urology, genetics, and psychoendrocrinology, is imperative when considering sex assignment and possible surgical repair. (134, 135)

The aim of surgical repair in females with ambiguous genitalia caused by CAH, when the decision is made by parents or patients themselves, is generally to remove the redundant erectile tissue, preserve the sexually sensitive glans clitoris, and provide a normal vaginal orifice that functions adequately for menstruation, intromission, and delivery. A medical indication for early surgery other than for sex assignment is recurrent urinary tract infections as a result of pooling of urine in the vagina or urogenital sinus.

Other Treatment Strategies

Glucocorticoid replacement has been an effective treatment for CAH for the past 50 years and remains its primary therapy; however, the management of these patients presents a challenge because inadequate treatment as well as oversuppression can both cause complications.

Bilateral adrenalectomy is a radical but effective measure in some cases. A few patients who were extremely difficult to control with medical therapy alone showed improvement in their symptoms after bilateral adrenalectomy (136). Because this approach renders the patient completely adrenal insufficient, however, it should be reserved for extreme cases and is not a good treatment option for patients who have a history of poor compliance with medication.

CONCLUSION

The pathophysiology of the various types of CAH (the most common being 21-OHD) can be traced to discrete, inherited defects in the genes encoding enzymes for adrenal steroidogenesis. Clinical presentation of each form is distinctive, depends largely on the underlying enzyme blockage, its precursor retention and deficient products. Treatment of CAH is targeted to replace the insufficient adrenal hormones. With proper hormone replacement therapy, normal and healthy development may often be expected. Glucocorticoid and, if necessary, mineralocorticoid replacement, has been the mainstay of treatment for CAH, but new treatment strategies continue to be developed and studied to improve care.